2-2--(hydroxynitrosohydrazono)bis-ethanamine and Hyperplasia

Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease.

Topics: Animals; Blotting, Western; Carotid Artery Injuries; Cell Cycle; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Hyperplasia; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Nitric Oxide; Nitric Oxide Donors; Rats; Rats, Sprague-Dawley; RNA Interference; Triazenes; Tunica Intima; Ubiquitin-Conjugating Enzymes; Ubiquitination

In this study the effects of nitric oxide (NO) on intimal hyperplasia (IH) were evaluated in an ex-vivo model of human saphenous vein (SV). SV segments were cultured in conditions able to reproduce IH (FCS), or in medium alone (RPMI), or in presence of a NO donor (NO). Osteopontin (OPN) and Interleukin (IL)-6 were determined in the medium at different culture times and in the tissue, at the end of experiment. OPN and IL-6 release in medium was increased in FCS with respect to RPMI (OPN: 13.9+/-2.9 vs. 2.3+/-0.8 microg/ml, p=0.0011; IL-6: 304.2+/-64.7 vs. 42.0+/-10.1 ng/ml, p<0.0006) as well as intima thickness, that positively correlated with OPN production (r=0.81). In tissue OPN was higher in FCS (82.0+/-30.3 ng/mg protein) than in RPMI (13.8+/-4.2, p=0.0051) and at baseline (3.7+/-0.7, p=0.018). NO reduces IH progression (25%) and both OPN and IL-6 expression (OPN/GAPDH: undetectable baseline; 0.27+/-0.06 RPMI; 0.89+/-0.28 FCS; 0.09+/-0.05 NO; p=0.026 FCS vs. baseline, p=0.018 vs. RPMI, p=0.005 vs. NO). The beneficial NO effect on IH reduction appears to be mediated by the indirect inhibition of OPN production. NO could modulates the initial inflammatory signals that induces the OPN over-production with the related cascade of events leading to IH.

Topics: Graft Occlusion, Vascular; Humans; Hyperplasia; Interleukin-6; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Osteopontin; Saphenous Vein; Stents; Tissue Culture Techniques; Tunica Intima

Type II diabetes mellitus (DM) and metabolic syndrome are associated with accelerated restenosis following vascular interventions due to neointimal hyperplasia. The efficacy of nitric oxide (NO)-based therapies is unknown in these environments. Therefore, the aim of this study is to examine the efficacy of NO in preventing neointimal hyperplasia in animal models of type II DM and metabolic syndrome and examine possible mechanisms for differences in outcomes. Aortic vascular smooth muscle cells (VSMC) were harvested from rodent models of type II DM (Zucker diabetic fatty), metabolic syndrome (obese Zucker), and their genetic control (lean Zucker). Interestingly, NO inhibited proliferation and induced G0/G1 cell cycle arrest to the greatest extent in VSMC from rodent models of metabolic syndrome and type II DM compared with controls. This heightened efficacy was associated with increased expression of cyclin-dependent kinase inhibitor p21, but not p27. Using the rat carotid artery injury model to assess the efficacy of NO in vivo, we found that the NO donor PROLI/NO inhibited neointimal hyperplasia to the greatest extent in type II DM rodents, followed by metabolic syndrome, then controls. Increased neointimal hyperplasia correlated with increased reactive oxygen species (ROS) production, as demonstrated by dihydroethidium staining, and NO inhibited this increase most in metabolic syndrome and DM. In conclusion, NO was surprisingly a more effective inhibitor of neointimal hyperplasia following arterial injury in type II DM and metabolic syndrome vs. control. This heightened efficacy may be secondary to greater inhibition of VSMC proliferation through cell cycle arrest and regulation of ROS expression, in addition to other possible unidentified mechanisms that deserve further exploration.

Topics: Animals; Aorta, Abdominal; Apoptosis; Carotid Artery Injuries; Cell Cycle; Cell Proliferation; Cells, Cultured; Diabetes Mellitus, Type 2; Disease Models, Animal; DNA Replication; Dose-Response Relationship, Drug; Hyperplasia; Male; Metabolic Syndrome; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Nitric Oxide; Nitric Oxide Donors; Proline; Rats; Rats, Zucker; Reactive Oxygen Species; S-Nitroso-N-Acetylpenicillamine; Triazenes; Tyrosine